Antioxidant combinations for use in feed rations to increase milk production and milk fat

ABSTRACT

The present invention provides a combination of antioxidants that effectively stabilize different types of fats utilized in a ruminant diet. When included in a ruminant feed ration or water source, the antioxidant combination typically increases nutrient digestion, such as fiber and protein, improves rumen fermentation, improves microbial growth, improves microbial efficiency, increases milk production and/or milk fat, improves antioxidant status of the ruminant, and attenuates the negative effects of some fats in the ruminant animal.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/807,152 filed on Jul. 12, 2006, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention generally provides combinations of antioxidantsfor use in ruminant feed rations. The antioxidant combinations, when fedto ruminant animals, reduce the formation of free radicals in the diet,improve nutrient digestion, and optimize fermentation in the rumen ofthe animal. In addition, the present invention also provides methods forincreasing milk production and/or milk fat, for improving theantioxidant status of the ruminant, and for reducing the negative rumeneffect associated with feeding a non-inert fat source to a ruminantanimal.

BACKGROUND OF THE INVENTION

Fats are concentrated sources of energy, and their addition to the feedrations of cattle and other ruminants has become standard practice.Fats, however, are prone to oxidation, a degradation process thatreduces their nutritional value and produces volatile compounds havingunpleasant smells and tastes (i.e., rancidity). The rate of oxidationincreases with the degree of unsaturation (or the number ofcarbon-carbon double bonds). During fat oxidation a free radical isformed by the removal of a labile hydrogen atom from a carbon atomadjacent to a double bond. The resultant free radical is susceptible toattack by oxygen to form a free radical peroxide, which then serves as acatalyst of further oxidation. Thus, the oxidative breakdown of fats isautocatalytic, giving rise to a chain reaction and the formation ofundesirable breakdown products.

The feeding of oxidized fats to ruminants, and dairy cows in particular,may contribute to the load of free radical in the animal and exacerbatethe susceptibility of the animal to oxidative stress. Furthermore, largeamounts of (oxidized or non oxidized) unsaturated or unsaturated fatscan interfere with the rumen microbial population, block fiberdegradation, and microbial growth. Because of the potential negativeimpact of certain fats, ruminally inert fats have been developed. Inertfats are fatty acids having increased saturation, fatty acids complexedwith calcium, or encapsulated fats. Regardless of how they are madeinert, however, inert fats are expensive.

Since feed is a major cost in ruminant production, it is desirable tosupplement their rations with lower cost non-inert fats, such asvegetable oils, blends of vegetable oils and animal fats, or feedingredients with a moderate to high content of fat, such as distillersgrains. These fats need to be stabilized, however. One way to stabilizeand inhibit the oxidation of fat sources used in ruminant diets is toinclude an antioxidant in the feed ration. One of the most effectiveantioxidants is ethoxyquin(6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, sold under the trademarkAGRADO®), which is widely used as an antioxidant or preservative in feedsupplements. While effective, ethoxyquin is generally more effective asan antioxidant for fish oil and animal fat, but not as effective forcontrolling oxidation of plant-derived oils. Moreover, dietaryantioxidants have traditionally been used only to control the oxidationof fat sources while they are in storage, but not to control theoxidation of the fat source once fed to a ruminant animal. Consequently,new antioxidant formulations that are effective at controlling oxidationof fats and lipids derived from a broad spectrum of fat sources remainsan unmet need in the art.

SUMMARY OF THE INVENTION

Among the various aspects of the invention, therefore, is a method forincreasing milk production and/or milk fat in a ruminant animal. Themethod comprises feeding to the ruminant animal a first antioxidant thatis a quinoline compound, and a second antioxidant that is different thanthe first antioxidant.

Other aspects of the invention will be in part apparent and in partpointed our hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the increased antioxidant protection providedto soybean oil by a blend of antioxidants. Plotted are the oil stability(OSI) induction times (square symbols) and the peroxide values (diamondsymbols) for soy oil without added antioxidants, soy oil with ethoxyquin(ETX), and soy oil with a blend of ethoxyquin and tertiary butylhydroquinone (ETX+TBHQ).

FIG. 2 is a graph showing the increased antioxidant protection providedto yellow grease by a blend of antioxidants. Plotted are the oilstability (OSI) induction times (square symbols) and the peroxide values(diamond symbols) for yellow grease without added antioxidants, yellowgrease with ethoxyquin (ETX), and yellow grease with a blend ofethoxyquin and tertiary butyl hydroquinone (ETX+TBHQ).

FIG. 3 is a graph illustrating the synergistic activity of twoantioxidants to stabilize wet distillers grains (WDG). Plotted are theoil stability (OSI) induction times for WDG without added antioxidants,WDG with a blend of ethoxyquin and tertiary butyl hydroquinone(ETX+TBHQ), WDG with ethoxyquin (ETX), and WDG with tertiary butylhydroquinone (TBHQ).

FIG. 4 is a graph illustrating the stabilization of omega-3 and omega-6fatty acids in the blended oil (a mix of corn oil, fish oil, and yellowgrease) by the antioxidants, ethoxyquin and tertiary butyl hydroquinone.Shown are the percentages of omega-3 fatty acids (solid line) andomega-6 fatty acids (bars) before oxidation (control) and afteroxidation in the absence of the antioxidants (oxidized) and afteroxidation in the presence of the antioxidants (A).

FIG. 5 is a graph illustrating the stabilization of eicosapentaenoicacid (EPA) and docosahexanenoic acid (DHA) in the blended oil (a mix ofcorn oil, fish oil, and yellow grease) by the antioxidants, ethoxyquinand tertiary butyl hydroquinone. Shown are the percentages of EPA (solidline) and DHA (bars) before oxidation (control) and after oxidation inthe absence of the antioxidants (oxidized) and after oxidation in thepresence of the antioxidants (A).

FIG. 6 are bar graphs illustrating the stabilization of oils in seeds bythe antioxidants, ethoxyquin and tertiary butyl hydroquinone. Presentedare the peroxide values in milliequivalents (meq) of peroxide per kg offat in grains without the antioxidants (gray bars) and grains with theantioxidants (black bars). Panel A presents the values for flax seed.Panel B presents the values for wet distillers grains.

FIG. 7 is a graph illustrating the stabilization of unsaturated fattyacids in wet distillers grains (WDG) by the antioxidants, ethyoxyquinand tertiary butyl hydroquinone. The percentages of linoleic acid C18:2(bars) and linolenic acid C18:3 (solid line) are shown.

DETAILED DESCRIPTION OF THE INVENTION

A combination of antioxidants has been discovered that effectivelyprevents the oxidation of the different types of fats (e.g., plantderived oils, blends of plant and other oils/fats, or distillers grains)utilized in a ruminant diet. In particular, the combination ofantioxidants prevents the oxidation of these fats more effectively thanthe summed activity of an equimolar amount of either antioxidant usedalone. It has also been discovered, as illustrated in the examples, thatthe antioxidant combinations of the invention are effective atattenuating the negative impact of dietary fat on rumen fermentation.Independent of the degree of oxidation of the dietary fat, theantioxidant combination improves nutrient digestion, fiber digestion,dry matter intake, antioxidant status, milk production and/or milk fatof a ruminant animal. Advantageously, the antioxidant combinations ofthe invention provide a means to feed fat sources, and in particularnon-inert fat sources, to a ruminant animal while attenuating thenegative rumen effects typically associated with feeding these fats to aruminant animal.

(I) Antioxidant Combinations

One aspect of the present invention provides antioxidant combinations ofat least two different antioxidants. The antioxidant combinations of theinvention may be formulated as a ruminant feed supplement or as apremix. Typically, the first antioxidant of the combination is aquinoline compound and the second antioxidant is different than thefirst antioxidant. Exemplary antioxidant combinations are formulated sothat the first antioxidant is more effective at reducing the oxidationof animal fat or fish fat compared to the second antioxidant and thesecond antioxidant is more effective at reducing the oxidation of plantfat, such as vegetable oils, compared to the first antioxidant. Byformulating the combination of antioxidants in this manner, a broadspectrum of fat sources, including fat sources relatively high inunsaturated fatty acids, may be utilized in the ruminant feed ration orwater source without negatively impacting rumen fermentation.

(a) First Antioxidant

The first antioxidant comprising the combination is a quinolinecompound. Typically, the quinoline compound will be a substituted1,2-dihydroquinoline. Substituted 1,2-dihydroquinoline compoundssuitable for use in the invention generally correspond to formula (I):

wherein:

-   -   R¹, R², R³ and R⁴ are independently selected from the group        consisting of hydrogen and an alkyl group having from 1 to about        6 carbons; and    -   R⁵ is an alkoxy group having from 1 to about 12 carbons.

In another embodiment, the substituted 1,2-dihydroquinoline will haveformula (I) wherein:

-   -   R¹, R², R³ and R⁴ are independently selected from the group        consisting of hydrogen and an alkyl group having from 1 to about        4 carbons; and    -   R⁵ is an alkoxy group having from 1 to about 4 carbons.

In one preferred embodiment, the substituted 1,2-dihydroquinoline willbe 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline having the formula:

The compound, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, commonlyknown as ethoxyquin, is sold under the trademark agrado®. The presentinvention also encompasses salts of ethoxyquin and other compoundshaving formula (i). Ethoxyquin and other compounds having formula (i)may be purchased commercially from novus international, inc. Or made inaccordance with methods generally known in the art, for example, asdetailed in U.S. Pat. No. 4,772,710, which is hereby incorporated byreference in its entirety.

(b) Second Antioxidant

The second antioxidant is different than the first antioxidant. Avariety of antioxidants are suitable for use in the antioxidantcombination of the present invention. Typically, the second antioxidantis not a surfactant. In some embodiments, the second antioxidant may bea compound that interrupts the free-radical chain of oxidative reactionsby protonating free radicals, thereby inactivating them. Alternatively,the second antioxidant may be a compound that scavenges the reactiveoxygen species. In other embodiments, the second antioxidant may be acompound that chelates the metal catalysts. In still other embodiments,the second antioxidant may be a synthetic compound, a semi-syntheticcompound, or a natural (or naturally-derived) compound.

Suitable antioxidants include, but are not limited to, ascorbic acid andits salts, ascorbyl palmitate, ascorbyl stearate, anoxomer,n-acetylcysteine, benzyl isothiocyanate, m-aminobenzoic acid,o-aminobenzoic acid, p-aminobenzoic acid (paba), butylatedhydroxyanisole (bha), butylated hydroxytoluene (bht), caffeic acid,canthaxantin, alpha-carotene, beta-carotene, beta-caraotene,beta-apo-carotenoic acid, carnosol, carvacrol, catechins, cetyl gallate,chlorogenic acid, citric acid and its salts, clove extract, coffee beanextract, p-coumaric acid, 3,4-dihydroxybenzoic acid,n,n′-diphenyl-p-phenylenediamine (dppd), dilauryl thiodipropionate,distearyl thiodipropionate, 2,6-di-tert-butylphenol, dodecyl gallate,edetic acid, ellagic acid, erythorbic acid, sodium erythorbate,esculetin, esculin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, ethylgallate, ethyl maltol, ethylenediaminetetraacetic acid (edta),eucalyptus extract, eugenol, ferulic acid, flavonoids (e.g., catechin,epicatechin, epicatechin gallate, epigallocatechin (egc),epigallocatechin gallate (egcg), polyphenol epigallocatechin-3-gallate),flavones (e.g., apigenin, chrysin, luteolin), flavonols (e.g.,datiscetin, myricetin, daemfero), flavanones, fraxetin, fumaric acid,gallic acid, gentian extract, gluconic acid, glycine, gum guaiacum,hesperetin, alpha-hydroxybenzyl phosphinic acid, hydroxycinammic acid,hydroxyglutaric acid, hydroquinone, n-hydroxysuccinic acid,hydroxytryrosol, hydroxyurea, rice bran extract, lactic acid and itssalts, lecithin, lecithin citrate; r-alpha-lipoic acid, lutein,lycopene, malic acid, maltol, 5-methoxy tryptamine, methyl gallate,monoglyceride citrate; monoisopropyl citrate; morin,beta-naphthoflavone, nordihydroguaiaretic acid (ndga), octyl gallate,oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine,phosphoric acid, phosphates, phytic acid, phytylubichromel, pimentoextract, propyl gallate, polyphosphates, quercetin, trans-resveratrol,rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin,sinapic acid, succinic acid, stearyl citrate, syringic acid, tartaricacid, thymol, tocopherols (i.e., alpha-, beta-, gamma- anddelta-tocopherol), tocotrienols (i.e., alpha-, beta-, gamma- anddelta-tocotrienols), tyrosol, vanilic acid,2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., ionox 100),2,4-(tris-3′,5′-bi-tert-butyl-4′-hydroxybenzyl)-mesitylene (i.e., ionox330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (tbhq), thiodipropionic acid, trihydroxy butyrophenone,tryptamine, tyramine, uric acid, vitamin k and derivates, vitamin q10,wheat germ oil, zeaxanthin, or combinations thereof.

Exemplary second antioxidants include synthetic phenolic compounds, suchas tbhq, bha, or bht; gallic acid derivatives, such as n-propyl gallate;vitamin c derivatives, such as ascorbyl palmitate; lecithin; and vitamine compounds, such as, alpha-tocopherol. In one preferred embodiment, thesecond antioxidant will be tbhq.

(c) Formulations of Antioxidant Combinations

Suitable antioxidant combinations for use in the present inventioninclude at least one of the quinoline compounds detailed in i (a) and atleast one of the second antioxidants detailed in i (b). In someembodiments, the combination may include only two differentantioxidants. In other embodiments, the combination may include at leastthree different antioxidants. In additional embodiments, the combinationmay include four or more different antioxidants. Non-limiting examplesof suitable antioxidant combinations are set-forth in table a (i.e., thefirst antioxidant in column one is combined with the second antioxidantin column two to form an antioxidant combination of the invention).

TABLE A First Antioxidant Second Antioxidant6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ascorbic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline an ascorbate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ascorbyl palmitate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ascorbyl stearate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline anoxomer6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline N-acetylcysteine6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline benzyl isothiocyanate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline m-aminobenzoic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline o-aminobenzoic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline p-aminobenzoic acid (PABA)6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline butylated hydroxyanisole(BHA) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline butylatedhydroxytoluene (BHT) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinecaffeic acid 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline canthaxantin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline alpha-carotene6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline beta-carotene6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline beta-caraotene6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline beta-apo-carotenoic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline carnosol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline carvacrol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a catechin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline cetyl gallate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline chlorogenic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline citric acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a citrate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline clove extract6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline coffee bean extract6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline p-coumaric acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline 3,4-dihydroxybenzoic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolineN,N′-diphenyl-p-phenylenediamine (DPPD)6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline dilauryl thiodipropionate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline distearyl thiodipropionate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline 2,6-di-tert-butylphenol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline dodecyl gallate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline edetic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ellagic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline erythorbic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline sodium erythorbate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline esculetin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline esculin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline6-ethoxy-1,2-dihydro-2,2,4- trimethylquinoline6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ethyl gallate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ethyl maltol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ethylenediaminetetraaceticacid (EDTA) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline eucalyptusextract 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline eugenol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ferulic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a flavonoid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline epigallocatechin (EGC)6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline epigallocatechin gallate(EGCG) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a flavone6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a flavonol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a flavanone6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline fraxetin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline fumaric acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline gallic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline gentian extract6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline gluconic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline glycine6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline gum guaiacum6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline hesperetin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline alpha-hydroxybenzylphosphinic acid 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinehydroxycinammic acid 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinehydroxyglutaric acid 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinehydroquinone 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolineN-hydroxysuccinic acid 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinehydroxytryrosol 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinehydroxyurea 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline lactic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline lactates6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline lecithin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline lecithin citrate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline R-alpha-lipoic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline lutein6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline lycopene6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline malic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline malates6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline maltol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline 5-methoxy tryptamine6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline methyl gallate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline monoglyceride citrate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline monoglyceride citrate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline morin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline beta-naphthoflavone6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline nordihydroguaiaretic acid(NDGA) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline octyl gallate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline oxalic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline an oxalate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline palmityl citrate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline phenothiazine6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline phosphatidylcholine6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline phosphoric acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a phosphate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline phytic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline phytylubichromel6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline pimento extract6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline propyl gallate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a polyphosphate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline quercetin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline trans-resveratrol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline rice bran extract6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline rosemary extract6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline rosmarinic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline sage extract6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline sesamol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline silymarin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline sinapic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline stearyl citrate6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline succinic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline syringic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline tartaric acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline tartrates6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline thymol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a tocopherol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline a tocotrienol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline tyrosol6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline vanilic acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline2,6-di-tert-butyl-4-hydroxymethyl phenol (i.e., Ionox 100)6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline2,4-(tris-3′,5′-bi-tert-butyl-4′- hydroxy benzyl)-mesitylene (i.e.,Ionox 330) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline2,4,5-trihydroxybutyrophenone6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline ubiquinone6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline tertiary butylhydroquinone (TBHQ) 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinethiodipropionic acid 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinetrihydroxy butyrophenone 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolinetryptamine 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline tyramine6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline uric acid6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline vitamin K6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline vitamin Q106-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline wheat germ oil6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline zeaxanthin6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline derivates of any of theforegoing

In one exemplary embodiment, the antioxidant combination is6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and any of the naturalantioxidants detailed herein. In a further exemplary embodiment, theantioxidant combination is 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinolineand BHA. In still another exemplary embodiment, the antioxidantcombination is 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and BHT. Ina further exemplary embodiment, the antioxidant combination is6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and TBHQ. As detailed inthe examples, the combination of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and TBHQ generallyprotects oils and fats from oxidation longer than the summed activity ofan equimolar amount of either antioxidant used alone. The combination of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and TBHQ is sold under thetrademark AGRADO PLUS®.

As will be appreciated by a skilled artisan the concentration of thefirst antioxidant and the concentration of the second antioxidantcomprising the antioxidant combination can and will vary depending uponthe particular antioxidants, the amount and type of fat source in thefeed ration, and the species and age of the ruminant animal that will befed the combination. By way of non-limiting example, when the ruminantis a beef cow, the amount of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline fed to the animal mayrange from about 50 to about 250 ppm, or from about 140 to about 160 ppmin its feed ration. In an exemplary embodiment, the amount of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline fed to a beef cow will be150 ppm. By way of further example, when the ruminant animal is a dairycow, the amount of 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline fed tothe animal may range from about 20 to about 250 ppm, or from about 55 toabout 75 ppm in its feed ration. In an exemplary embodiment, the amountof 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline fed to a dairy cow willbe 65 ppm. Other exemplary formulations of antioxidant combinations aredetailed in sections I (d), (e), and in the examples.

(d) Liquid Compositions

The antioxidant combinations of the invention, when formulated as acomposition, may be a liquid composition or a dry composition. Forembodiments where the antioxidant combination comprises a liquidcomposition, the composition will typically include a solvent carrierselected from a polar solvent, a non-polar solvent, or combinations ofboth.

Generally speaking, a polar solvent may be used when an antioxidant inthe combination is a water-soluble antioxidant. Suitable examples ofpolar solvents include, but are not limited to, glycerol, isopropylalcohol, ethyl alcohol, propylene glycol, erythritol, xylitol, sorbitol,maltitol, mannitol, water, or mixtures thereof. In one embodiment thepolar solvent may be glycerol. The concentration of the polar solventwill vary depending upon the combination of antioxidants in thecomposition. In general, the percent by volume of the polar solvent mayrange from about 5% to about 50%. The percent by volume of polar solventmay be about 5%, 10%, 15%, 20%, or 25%.

The liquid composition may also include a nonpolar solvent. In general,a nonpolar solvent may be used when an antioxidant in the combination isa lipid-soluble antioxidants. Suitable examples of nonpolar solventsinclude, but are not limited to, monoglycerides, diglycerides, vegetableoil, or combinations thereof. The monoglycerides and diglycerides may bedistilled from vegetable oils or they may be synthesized via anesterification reaction. The vegetable oil may be corn oil, soybean oil,canola oil, cottonseed oil, palm oil, peanut oil, safflower oil, andsunflower oil. In one embodiment, the nonpolar solvent may be corn oil.In another embodiment, the nonpolar solvent may comprise monoglyceridesand corn oil. The concentration of the nonpolar solvent will varydepending upon the combination of antioxidants in the composition. Ingeneral, the percent by volume of the nonpolar solvent may range fromabout 5% to about 50%. The percent by volume of the nonpolar solvent maybe 10%, 15%, 20%, or 25%.

By way of non-limiting example, a liquid composition of the inventionmay comprise from about 40% to about 75% by weight of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, from about 1% to about20% by weight of tertiary butyl hydroquinone, and from about 10% toabout 30% by weight of at least one solvent carrier. In anotherembodiment, the liquid composition may comprise from about 60% to about70% by weight of 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, fromabout 1% to about 10% by weight of tertiary butyl hydroquinone, and fromabout 10% to about 30% by weight of at least one solvent carrier. In anexemplary embodiment, the liquid composition consists of about 65% byweight 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, about 7% by weighttertiary butyl hydroquinone, about 7% by weight citric acid, about 19%by weight propylene glycol, and about 2% by weight corn oil.

(e) Dry Compositions

Alternatively, the antioxidant combination may be formulated as a drycomposition. Typically, when formulated as a dry composition, one ormore carriers may be utilized. In an exemplary embodiment, the drycomposition will be flowable. In this context, “flowable” means that thedry composition is substantially free flowing and substantiallyresistant to clumping.

Several inorganic carriers are suitable for use in the present inventionto formulate a dry composition of antioxidants. The inorganic carrierwill typically be granular, it may be porous, and is generallybiologically inert. In this context, an inorganic carrier isbiologically inert if it is nontoxic and does not generate anappreciable immune reaction when administered to an animal. Non-limitingexamples of suitable inorganic carriers include natural or regeneratedmineral substrates. One preferred class of mineral carriers is thesilicate class. The silicate utilized in the present invention may beselected from a silicate subclass selected from the group consisting ofnesosilicate, sorosilicate, inosilicate, cyclosilicate, phyllosilicateand tectosilicate. Examples of suitable nesosilicates include aluminumsilicate, iron magnesium manganese aluminum silicate hydroxide, calciumboro-silicate hydroxide, beryllium aluminum silicate hydroxide, ironsilicate, magnesium silicate, yttrium iron beryllium silicate, ironaluminum silicate, calcium iron silicate, calcium aluminum silicate,magnesium aluminum silicate, calcium chromium silicate, calciumboro-silicate hydroxide, aluminum silicate, magnesium iron silicate,berylium silicate, calcium titanium silicate, zinc silicate andzirconium silicate. Suitable examples of sorosilicates include berylliumsilicate hydroxide, calcium boro-silicate, yttrium cerium calciumaluminum iron silicate hydroxide, calcium aluminum silicate hydroxide,calcium iron aluminum silicate hydroxide, calcium aluminum silicatehydroxide, and calcium iron silicate hydroxide. Non-limiting examples ofsuitable inosilicates include sodium titanium silicate, calciumsilicate, sodium iron silicate, calcium sodium magnesium aluminum irontitanium silicate, calcium magnesium silicate, magnesium silicate,calcium iron silicate, magnesium iron silicate, sodium aluminum ironsilicate, lithium aluminum silicate, manganese iron magnesium calciumsilicate, sodium manganese calcium silicate hydroxide, copper silicatehydroxide, calcium silicate, calcium magnesium iron silicate hydroxide,magnesium iron silicate hydroxide, iron magnesium silicate hydroxide,potassium iron titanium silicate hydroxide, and calcium iron manganesesilicate hydroxide. Suitable examples of cyclosilicates include calciummagnesium iron manganese aluminum borosilicate, potassium lithiumcalcium titanium zirconium silicate, barium titanium silicate, berylliumaluminum silicate, magnesium aluminum silicate, potassium sodium ironmagnesium aluminum silicate, sodium magnesium aluminum boro-silicatehydroxide, and potassium sodium lithium iron manganese aluminumsilicate. Examples of suitable phyllosilicates include hydratedpotassium sodium calcium silicate, hydrated calcium vanadium silicate,hydrated copper aluminum hydrogen silicate hydroxide, iron magnesiumaluminum silicate hydroxide, iron magnesium aluminum silicate hydroxide,lithium aluminum silicate hydroxide, aluminum silicate hydroxide,magnesium silicate hydroxide, hydrated calcium silicate hydroxide,potassium iron magnesium aluminum silicate hydroxide fluoride, potassiumlithium aluminum silicate hydroxide fluoride, potassium aluminumsilicate hydroxide fluoride, potassium magnesium aluminum silicatehydroxide fluoride, calcium aluminum silicate hydroxide, and ironmagnesium silicate hydroxide. Suitable examples of tectosilicatesinclude sodium aluminum silicate, sodium calcium aluminum silicate,calcium aluminum silicate, calcium sodium aluminum silicate, sodiumcalcium aluminum silicate, potassium aluminum silicate, sodium calciumsilicate, silicon dioxide, sodium calcium aluminum silicate carbonate,sodium calcium aluminum silicate sulfate sulfide chloride, sodiumaluminum silicate chloride, calcium sodium aluminum silicate chloridecarbonate sulfate, hydrated sodium aluminum silicate, hydrated calciumaluminum silicate, hydrated barium potassium aluminum silicate, andhydrated sodium calcium aluminum silicate. In a preferred embodiment,the inorganic substrate is silicon dioxide or sodium benetonite.Depending upon the embodiment, the inorganic carrier may be a mixture ofcompounds, such as a mixture of one or more of any of the aforementionedsilicates.

It will be appreciated by those of skill in the art that the particlesize of the inorganic carrier as well as the concentration of inorganiccarrier can and will vary. In general, the average particle size ofinorganic carrier may be from about 50 microns to about 1000 microns. Inanother embodiment, the average particle size of the inorganic carriermay be from about 100 microns to about 500 microns. In yet anotherembodiment, the average particle size of the inorganic carrier may befrom about 100 microns to about 200 microns. In another embodiment, theconcentration of inorganic carrier included in the dry composition maybe from about 0.1% to about 0.5% by weight dm of the dry composition. Instill another embodiment, the concentration of inorganic carrierincluded in the dry composition may be from about 1.0% to about 5.0% byweight dm of the dry composition. In yet another embodiment, theconcentration of inorganic carrier included in the dry composition maybe from about 2.5% to about 15.0% by weight dm of the dry composition.

By way of non limiting example, a dry composition of the invention maycomprise from about 30% to about 70% by weight dm of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, from about 1% to about10% by weight dm of tertiary butyl hydroquinone, and from about 0.1% toabout 15% by weight dm of a carrier. In another embodiment, the drycomposition may comprise from about 45% to about 55% by weight dm of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, from about 3% to about 7%by weight dm of tertiary butyl hydroquinone, and from about 0.1% toabout 15% by weight dm of a carrier. In an exemplary embodiment, the drycomposition consists of about 50% by weight dm of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, about 5% by weight dm oftertiary butyl hydroquinone, about 5% by weight dm of citric acid, andabout 10% by weight dm of calcium carbonate.

(II) Feed Pre-Mixes or Supplements

Another aspect of the invention comprises a ruminant animal feed premixor feed supplement comprising the antioxidant combinations of theinvention. Typically, the premix will be added to various formulationsof grain concentrates and forages to formulate a ruminant animal feedration. As will be appreciated by the skilled artisan, the particularpremix formulation can and will vary depending upon the feed ration andanimal that the feed ration will be fed to. In addition to theantioxidant combination of the invention, the premix may furtheroptionally include one or more of a mixture of natural amino acids,analogs of natural amino acids, such as a hydroxyl analog of methionine(“HMTBA”), supplemental protein, supplemental fat, vitamins andderivatives thereof, enzymes, animal drugs, hormones, effectivemicroorganisms, organic acids, preservatives, and flavors.

In one embodiment, the feed premix may include one or more amino acids.Suitable examples of amino acids, depending upon the formulation,include alanine, arginine, asparagines, aspartate, cysteine, glutamate,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. Other amino acids usable as feed additives include, by way ofnon-limiting example, N-acylamino acids, hydroxy homologue compounds,and physiologically acceptable salts thereof, such as hydrochlorides,hydrosulfates, ammonium salts, potassium salts, calcium salts, magnesiumsalts and sodium salts of amino acids.

In one preferred embodiment, the antioxidant combination may be combinedwith a hydroxy analog of methionine (“HMTBA”) to form a feed pre-mix.Suitable hydroxyl analogs of methionine include2-hydroxy-4(methylthio)butanoic acid (sold by Novus International, St.Louis, Mo. under the trademark ALIMET®), its salts, esters, amides, andoligomers. Representative salts of HMTBA include the ammonium salt, thestoichiometric and hyperstoichiometric alkaline earth metal salts (e.g.,magnesium and calcium), the stoichiometric and hyperstoichiometricalkali metal salts (e.g., lithium, sodium, and potassium), and thestoichiometric and hyperstoichiometric zinc salt. Representative estersof HMTBA include the methyl, ethyl, 2-propyl, butyl, and 3-methylbutylesters of HMTBA. Representative amides of HMTBA include methylamide,dimethylamide, ethylmethylamide, butylamide, dibutylamide, andbutylmethylamide. Representative oligomers of HMTBA include its dimers,trimers, tetramers and oligomers that include a greater number ofrepeating units.

In still another embodiment, the feed premix will include supplementalprotein. Examples of supplemental protein include soybean meal, poultryblood meal, fish meal, meat meal, and crude soybean protein.

In yet another embodiment, the feed premix will include a fat source.The fat source may be a non-inert fat. The fat source may be a non-inertfat. Non-limiting examples of non-inert fats include plant derived oils(e.g., canola oil, corn oil, cottonseed oil, palm oil, peanut oil,safflower oil, soybean oil, and sunflower oil), fish oils (e.g.,menhaden oil, anchovy oil, albacore tuna oil, cod liver oil, herringoil, lake trout oil, mackerel oil, salmon oil, and sardine oil), animalfats (e.g., poultry fat, beef tallow, butter, pork lard, and whaleblubber), yellow grease (i.e., waste grease from restaurants andlow-grade fats from rendering plants), and combinations thereof. Thenon-inert fat source may also be a high fat product such as fish meal(e.g., menhaden meal, anchovy meal, herring meal, pollack meal, salmonmeal, tuna meal, and whitefish meal), oilseeds (e.g., canola seeds,cottonseeds, flax seeds, linseeds, Niger seeds, sesame seeds, soy beans,and sunflower seeds), or distillers grains (e.g., dried distillersgrains and solubles (DDGS) and wet distillers grains). The fat sourcemay be a ruminally inert fat. Suitable examples of ruminally inert fatsinclude calcium salts of palm fatty acids (e.g., MEGALAC®), saturatedfree fatty acids (e.g., Energy Booster 100), or hydrogenated tallow(e.g., ALIFET®). Some commercially available bypass fats are described,for example, in U.S. Pat. Nos. 5,182,126; 5,250,307; 5,391,787;5,425,963; and 5,456,927 which disclose C14-C22 fatty acids, theirglycerides, or their salts including, but not limited to, palmitic,oleic, linoleic, stearic, and lauric compounds.

In still another embodiment, the feed premix will include vitamins orderivatives of vitamins. Examples of suitable vitamins and derivativesthereof include vitamin A, vitamin A palmitate, vitamin A acetate,β-carotene, vitamin D (e.g., D₂, D₃, and D₄), vitamin E, menadionesodium bisulfite, vitamin B (e.g., thiamin, thiamin hydrochloride,riboflavin, nicotinic acid, nicotinic amide, calcium pantothenate,pantothenate choline, pyridoxine hydrochloride, cyanocobalamin, biotin,folic acid, p-aminobenzoic acid), vitamin K, vitamin Q, vitamin F, andvitamin C.

In yet another embodiment, the feed premix will include one or moreenzymes. Suitable examples of enzymes include protease, amylase, lipase,cellulase, xylanase, pectinase, phytase, hemicellulase and otherphysiologically effective enzymes.

In still another embodiment, the feed premix will include a drugapproved for use in ruminant animals. Non-limiting examples of suitableanimal drugs include antibiotics such as tetracycline type (e.g.,chlortetracycline and oxytetracycline), amino sugar type, ionophores(e.g., rumensin, virginiamycin, and bambermycin) and macrolide typeantibiotics.

In an additional embodiment, the feed premix will include a hormone.Suitable hormones include estrogen, stilbestrol, hexestrol, tyroprotein,glucocorticoids, insulin, glucagon, gastrin, calcitonin, somatotropin,and goitradien.

In a further embodiment, the feed premix will include an effectivemicroorganism. Examples of suitable effective microorganisms includelive and dead yeast cultures, which may be formulated as a probiotic. Byway of example, such yeast cultures may include one or more ofLactobacillus Acidophilus, Bifedobact Thermophilum, Bifedobat Longhum,Streptococcus Faecium, Sacchromyces cerevisiae, Bacillus pumilus,Bacillus subtilis, Bacillus licheniformis, Lactobacillus acidophilus,Lactobacillus casei, Enterococcus faecium, Bifidobacterium bifidium,Propionibacterium acidipropionici, Propionibacteriium freudenreichii,Aspergillus oryzae, and Bifidobacterium Pscudolongum.

In yet another embodiment, the premix will include an organic acid.Suitable organic acids include malic acid, propionic acid and fumaricacid.

In an additional embodiment, the feed premix will include a substance toincrease the palatability of the feed ration. Suitable examples of suchsubstances include natural sweeteners, such as molasses, and artificialsweeteners such as saccharin and aspartame.

As will be appreciated by the skilled artisan, any of the substance thatmay be included in the premix of the invention can be used alone or incombination with one another. The concentration of these additives willdepend upon the application but, in general, will be between about0.0001% and about 10% by weight of the dry matter, more preferablybetween about 0.001% and about 7.5%, most preferably between about 0.01%and about 5%.

(III) Animal Feed Rations and Water Sources

A further aspect of the invention encompasses an animal feed ration or awater source comprising the antioxidant combination or a premixcontaining the antioxidant combination. The feed ration may beformulated to meet the nutritional requirements of a variety ofruminants. Examples of typical feed rations for a variety of ruminantsare detailed below by way of non-limiting example.

(a) Feed Ingredients

Feed ingredients that may be utilized in the present invention tosatisfy a ruminant's maintenance energy requirements may include feedingredients that are commonly provided to ruminants for consumption.Examples of such feed ingredients include forage, grain, feed meals,feed concentrates, vitamins, minerals, and the like. Because theantioxidant combinations of the invention are effective in reducingoxidation of both animal or fish derived fats and plant derived fats, avariety of fat sources may be utilized in a typical ration of theinvention.

Forage products are feed ingredients such as vegetative plants in eithera fresh (pasture grass or vegetation), dried, or ensiled state and mayincidentally include minor proportions of grain (e.g., kernels of cornthat remain in harvested corn plant material after harvest). Forageincludes plants that have been harvested and optionally fermented priorto being provided to ruminants as a part of the feed of the presentinvention. Thus, forage includes hay, haylage, and silage. Examples ofhay include harvested grass, either indigenous to the location of theruminants being fed or shipped to the feeding location from a remotelocation. Non-limiting examples of hay include alfalfa, Bermuda grass,bahia grass, limpo grass, rye grass, wheat grass, fescue, clover, andthe like as well as other grass varieties that may be native to thelocation of the ruminants being provided the ruminant feed ration.

It is beneficial if the forage is relatively high quality (i.e.,contains relatively levels of metabolizable nutrients which permit theruminant to satisfy its nutrient and maintenance energy requirementsbefore reaching its consumption capacity). If the forage is of lowquality, the ruminant may not metabolize it adequately to achievedesired performance effects (e.g., satisfy its nutrient and/ormaintenance energy requirements), not only compromising the nutritionalbenefit from the forage per se, but also causing the ruminant to feelfull or bloated, and possibly deterring it from consuming sufficientnutrients.

Haylage is a forage product that has been naturally fermented byharvesting a hay crop while the sap is still in the plant. The harvestedhay or hay bales are then stored in an air-tight manner in whichfermentation can occur. The fermentation process converts the sugars inthe plants into acids which lower the pH of the harvested hay andpreserves the forage.

Silage, similar to haylage, is a forage product that is produced fromthe harvest, storage and fermentation of green forage crops such as cornand grain sorghum plants. These crops are chopped, stems and all, beforethe grain is ready for harvest. The plant material is stored in silos,storage bags, bunkers or covered piles causing the material to ferment,thereby lowering the pH and preserving the plant material until it canbe fed.

Forage products also include high fiber sources and scrap vegetationproducts such as green chop, corncobs, plant stalks, and the like.

Grain products include corn, corn gluten meal, soybeans, soybean meal,wheat, barley, oats, sorghum, rye, rice, and other grains and grainmeals.

Feed concentrates are ruminant feedstuffs that are high in energy andlow in crude fiber. Concentrates also include a source of one or moreingredients that are used to enhance the nutritional adequacy of a feedsupplement mix, such as vitamins and minerals.

The fat source may be a non-inert fat, such as plant oils, fish oils,animal fats, yellow grease, fish meal, oilseeds, distillers grains, orcombinations thereof. Non-limiting examples of non-inert fats werepresented above in Section II. The fat source may also be a ruminallyinert fat, as described above in Section II. The fat source willgenerally comprise from about 1% to about 10% of the dry mass of thetotal feed ration, more preferably from about 2% to about 6%, and mostpreferably from about 3% to about 4%.

Other ingredients may be optionally included in the ruminant feed toprovide additional nutrients to the ruminants. Examples of optionalingredients include urea, vitamins, minerals, and the like. Ureaprovides rumen bacteria a source of non-protein nitrogen from which theyare able to synthesize bacterial protein. These ingredients may also beexcluded as necessary to provide a feed ration to ruminants that can betailored to meet their nutritional needs.

(b) Feed Rations

Feed rations of the present invention typically are formulated to meetthe nutrient and energy demands of a particular ruminant animal. Thenutrient and energy content of many common ruminant feed ingredientshave been measured and are available to the public. The NationalResearch Council has published books that contain tables of commonruminant feed ingredients and their respective measured nutrient andenergy content. Additionally, estimates of nutrient and maintenanceenergy requirements are provided for growing and finishing cattleaccording to the weight of the cattle. National Academy of Sciences,Nutrient Requirements of Beef Cattle, Appendix Tables 1-19, 192-214,(National Academy Press, 2000); Nutrient Requirements of Dairy Cattle(2001), which are each incorporated herein by their entirety. Thisinformation can be utilized by one skilled in the art to estimate thenutritional and maintenance energy requirements of growing cattle ordairy cattle and determine the nutrient and energy content of ruminantfeed ingredients.

In one embodiment, the feed ration will be formulated for a dairy cow.In practice, ruminants are typically fed as a ration, commonly referredto as a total mixed ration (TMR), which consists of a forage portion anda grain concentrate portion. Any of the forage and grain concentratesdetailed herein or otherwise known in the art may be utilized. As willbe appreciated by a skilled artisan, a feed ration for a dairy cow canand will vary greatly depending upon the cow's stage of production. Inthis context, stage of production not only refers to whether a dairy cowis dry or lactating, but also the duration of time the cow has been inthe dry cycle or the lactation cycle. Milestones in the stage ofproduction include the first 35 days dry, known as “far off;” the last21 days dry, known as “close-up;” day 0 to day 14 of lactation, known as“fresh;” day 14 to day 80 of lactation, known as “peak milk;” days 80 to200 of lactation, known as “peak intake;” and days 200 to 330 oflactation. Suitable rations for dairy cattle for the first 35 days dry,day 0 to 14 of lactation and day 14 to 80 of lactation are detailedbelow.

An example of a suitable dairy cow feed ration for a cow in the first 35days of the dry cycle is as follows:

Percent by Weight (DM basis) Ingredient of Total Feed CompositionSteamrolled Corn 8.0 Wheat straw 8.5 Alfalfa hay 38.0 Corn silage 41.0Trace Mineral Salts 4.5

A suitable example of a dairy cow feed ration for a cow at day 0 to 14of the lactation cycle is as follows:

Percent by Weight (DM basis) Ingredient of Total Feed CompositionSteamrolled Corn 8.0 Soybean meal (44%) 7.5 Alfalfa hay 17.0 Corn silage47.0 Trace Mineral Salts 4.5

An example of a suitable dairy cow feed ration for a cow at day 14 to 80of the lactation cycle is as follows:

Percent by Weight (DM basis) Ingredient of Total Feed CompositionSteamrolled Corn 15.0 Soybean meal (44%) 13.0 Alfalfa hay 22.0 Cornsilage 21.0 Distillers grains 8.0 Whole Cottonseed 10.0 Soyean hulls 6.5Trace Mineral Salts 4.5

A feed ration may also be formulated to meet the nutritionalrequirements of non-dairy cattle, and in particular, feedlot cattle. Thepercentage of each type of component in the cattle diet (i.e. grain toroughage ratio) depends upon the dietary requirements of the particularanimal. By way of example, a feed composition typically fed to feedlotcattle on an intermediate or growing diet may include:

Percent by Weight of Ingredient Total Feed Composition DehydratedAlfalfa Meal 25.0 Cottonseed Hulls 5.0 Steamrolled Corn 60.0 Soybeanmeal (44%) 3.0 Calcium Carbonate 1.0 Sodium Tripolyphosphate 0.5 CaneMolasses 5.0 Trace Mineral Salts 0.5

The intermediate diet contains a moderate energy to roughage ratio andis fed to cattle during their growth stage. After the intermediate diet,a higher energy finishing diet is substituted until the cattle are readyfor slaughter. A typical finishing diet may include:

Percent by Weight of Ingredient Total Feed Composition DehydratedAlfalfa Meal 5.0 Cottonseed Hulls 10.0 Steamrolled Corn 74.8 Soybeanmeal (44%) 3.0 Calcium Carbonate 0.7 Sodium Tripolyphosphate 0.3 CaneMolasses 5.0

All publications, patents, patent applications and other referencescited in this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor other reference were specifically and individually indicated to beincorporated by reference.

(IV) Method for Increasing Nutrient Digestion and Improving RuminantPerformance

Yet another aspect of the invention encompasses methods of using thecompositions of the invention for feeding a ruminant animal. Inparticular, methods for increasing nutrient digestion or performanceparameters of a ruminant animal are provided. The method comprisesfeeding the antioxidant combination, as described in Section (I), or apremix containing the antioxidant combination, as described in Section(II), to an animal that has been fed or may be fed a fat source. In anexemplary embodiment, the fat source comprises a non-inert fat source.

Providing the antioxidant combination to a ruminant may lead toincreased rumen fermentation. Increased rumen fermentation may lead toincreased fiber digestion, increased protein digestion, and improvedmicrobial growth and efficiency. Improved fiber digestion or degradationmay be an indication that addition of the antioxidant combinationreduces the negative effects associated with including non-inert fat inthe rumen diet. In this context, the antioxidant combinations of theinvention, when fed in a feed ration to a ruminant, provide a means forincluding higher amounts of non-inert fats in the diet while attenuatingthe negative rumen effects typically observed by feeding this type offat. Alternatively, in certain embodiments it may be possible to totallyreplace inert fat with non-inert fat in the Feeding the antioxidantcombination may also improve dry matter intake (DMI). Furthermore,providing the antioxidant combination may also improve the antioxidantstatus of the animal, whereby the animal is less susceptible tooxidative stress. Feeding the antioxidant combination to a lactatingbeef cow may also increase milk yield, milk fat and fat corrected milk.

DEFINITIONS

“DM” is an abbreviation for dry matter.

The term “fat source,” as used herein, refers to a molecule containingat least one lipid.

The term “improved antioxidant status” as used herein, refers to animproved antioxidant capacity of the animal to remove free radicals fromits system.

The term “lipids,” as used herein, refers to a substance that is waterinsoluble, but soluble in organic solvents (e.g., ether, chloroform,hexane, etc.). One example of a simple lipid is triglycerides.Triglycerides are found primarily in cereal grains, oilseeds and animalfats. The basic structure of triglycerides consists of one unit ofglycerol and three units of fatty acids.

The term “negative rumen effect” as used herein, refers to the toxiceffect that dietary fat and fatty acids have on the ruminal microfloraby reducing their growth and activity, which can result in, for example,reduced fiber and protein digestion.

The term “nutrient,” as used herein, refers to chemical substances thatare generally necessary for one or more of the maintenance, growth,production, reproduction and/or health of the ruminant. By way ofnon-limiting example, nutrients include water, energy (e.g.,carbohydrates, proteins, and lipids), proteins (e.g., nitrogenouscompounds), minerals, and vitamins.

Ppm stands for parts per million.

The term “ruminant” when used herein is meant to encompass mature andimmature animals with multi-compartment stomachs, including but notlimited to, cattle, sheep, deer, goats, musk, ox, buffalo, giraffe andcamels. For example, cattle and sheep have a stomach with fourcompartments comprising the rumen, reticulum, omasum and abomasum.

As various changes could be made in the above compounds, products andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

EXAMPLES

The following non-limiting examples illustrate various embodiments ofthe invention.

Example 1 Effectiveness of antioxidant blends vs. Individualantioxidants in Stabilizing Oils or Fats

The purpose of this study was to compare the effectiveness of a blend ofethoxyquin (ETX) and tertiary butyl hydroquinone (TBHQ) versus ETX aloneor TBHQ alone to prevent the oxidation of soybean oil, yellow grease,and wet distiller grains. A combination of ETX and TBHQ is sold underthe trademark AGRADO PLUS® (Novus International, Inc.; St. Louis, Mo.).AGRADO PLUS® is a blend of 65% ethoxyquin, 7% tertiary butylhydroquinone, 7% citric acid, 19% propylene glycol, and 2% corn oil.Preparations of ETX are sold under the trademarks AGRADO® and SANTOQUIN®(Novus International, Inc.; St. Louis, Mo.).

Methods. The stability of the lipid material was assessed using theactive oxygen method (AOM) and/or the oil stability index (OSI) methods.The AOM measures the stability of an oil or fat by bubbling air throughthe oil or fat sample using specific conditions of flow rate,temperature, and concentration. The temperature used for theseexperiments was 98° C. At intervals, peroxides and hydroperoxidesproduced by this treatment were determined by titration with iodine. TheAOM value was defined as the milliequivalents of peroxide per kg of fat(meq/kg) after 20 hours. The lower the number, the more stable the oilor fat.

The OSI method is similar to the AOM method in that air is passedthrough a sample held at constant temperature. The temperature used forthese experiments was 110° C. After the air passed through the sample,it was bubbled through a reservoir of deionized water. Volatile acidsproduced by lipid oxidation dissolved in the water and increased itsconductivity. Conductivity of the water was monitored continuously andthe OSI value was defined as the hours required for the rate ofconductivity change to reach a predetermined value. The higher the OSIvalue, the more stable the oil or fat.

Results. The oxidative stability of soybean oil was assessed in theabsence of antioxidants (control) or in the presence of 500 ppm of ETXor 500 ppm of ETX+TBHQ. As shown in FIG. 1, the combination of ETX+TBHQincreased the OSI induction time to 12 hr, whereas the control and ETXalone had induction times of about 6 hr. Likewise, soybean oil subjectedto oxidative stress in the presence of ETX+TBHQ had much lower peroxidevalues (near 0) than soybean oil stressed in the presence of ETX alone(about 11 meq).

FIG. 2 presents similar data showing increased OSI induction time andreduced AOM peroxide levels in yellow grease stressed in the presence of500 ppm of ETX+TBHQ versus 500 ppm of ETX alone. Wet distillers grainswere subjected to oxidative stress in the presence of 500 ppm of ETX,100 ppm of TBHQ, and a blend of 500 ppm of ETX+100 ppm of TBHQ. FIG. 3illustrates the synergistic stabilizing effect of the combination ofETX+TBHQ. The OSI induction time of distillers grains treated with thecombination was much greater than the summed OSI induction times of ETXalone and TBHQ alone.

These data reveal that a combination of ethoxyquin and TBHQ is much moreeffective at stabilizing lipid materials than ethoxyquin used alone.

Example 2 The Combination of Ethoxyquin and Tertiary Butyl HydroquinoneStabilize Oils and Fats Used in Ruminant Diets

The objective of this study was to evaluate the ability of a combinationof ethoxyquin and tbhq to stabilize non-inert oils and fats used incattle diets.

Methods. The degree of oxidation and the fatty acid profile weredetermined in five different sources of dietary oils or fats before andafter artificial oxidative stresses. The oils or fats were: corn oil(co), soybean oil (so); menhaden fish oil (fo); yellow grease (yg); anda blend of corn oil, fish oil, and yellow grease (bo). The quality ofeach oil or fat was determined initially by determining the degree ofoxidation (initial peroxide value, ipv). The initial peroxide value is ameasure of the concentration of peroxides and hydroperoxides formedduring the initial stages of lipid oxidation. Milliequivalents ofperoxide per kg of fat (meq/kg) were measured by titration with iodideion.

The oils or fats were subjected to artificial oxidative stresses in theabsence or presence of 500 ppm of agrado plus® (see example 1). Thestability of the oils or fats was assessed using the aom and osimethods, as detailed in example 1. The temperatures used during aom were55° C. for the menhaden fish oil and 98° C. for the other oils and fats.The temperatures used during the osi method were 70° C. for the menhadenfish oil and 110° c. for the other oils and fats. Each oil or fat samplewas run in triplicate.

Fatty acid profiles were determined on the fats or oils before and afterthe 20 hr aom artificial stress in the absence or presence of theantioxidants. The fatty acid profile (fap) was performed on a hp5890a gc(agilent technologies, inc.; Santa Clara, Calif.) equipped with a flameionization detector and a 30 m×0.25 mm (0.2 μm film) supelco 2380 fusedsilica capillary column. The injector and detector temperatures wereheld at 250° C. and 260° C., respectively. The carrier gas was he (20cm/s) with an inlet pressure of 104 kpa. The column temperature wasprogrammed for 140° C. for 3 min, then increased to 220° C. at 2°C./min, and held at 220° C. for 2 min. Peaks were quantified bycomparison to an internal standard (c17:0).

Results. The high initial peroxide levels for the blended oil, menhadenfish oil and yellow grease (data not shown) revealed that these lipidswere subject to oxidation prior to any artificial stress applied. Alllipid sources were readily oxidized under the artificial environment(table 1). In the presence of the blend of antioxidants, all of thelipid sources exhibited a significant reduction in aom values after 20hours in the artificial environment (table 1). Although the antioxidantssignificantly reduced the oxidation in the blended oil (bo) sample, theaom value was still above the threshold of 20 meq/kg, which is thegenerally accepted threshold of rancidity. It appears that the blendedoil preparation presents a greater challenge to stabilization than anyof the homogenous oil or fat sources. The osi values were significantlyincreased for all of the oils and fats tested (table 1). The valuesranged from an increase of 3 hours for the blended oil to an increase of41.9 hours for the fish oil.

TABLE 1 Mean active oxygen method and oil stability index values afteroxidative stress. ETX + TBHQ Control LSMSE Active Oxygen Method 20 h(meq/kg fat) Corn Oil 4.7^(a) 120.6^(b) 8.3 Blended Oil 106.6^(a)138.9^(b) Fish Oil 3.9^(a) 252.6^(b) Soybean Oil 11.8^(a) 237.3^(b)Yellow Grease 17.1^(a) 238.4^(b) Oil Stability Index (hours) Corn Oil17.2^(a) 8.1^(b) 1.2 Blended Oil 3.7^(a) 0.7^(b) Fish Oil 44.7^(a)2.8^(b) Soybean Oil 15.2^(a) 6.3^(b) Yellow Grease 11.7^(a) 4.3^(b)^(a,b)Means with different letters within a row are significantlydifferent (P < 0.05).

Oxidization resulted in a significant modification of the fatty acidprofile of the tested oils and fats. The concentration of linoleic acid(C18:2) and linolenic acid (C18:3) for the blended oil, soybean oil, andyellow grease was maintained in the presence of the antioxidants,whereas the oxidized lipids had a significant decrease in these fattyacids (Table 2). There was also a significant increase in theconcentration of palmitic acid (C16:0) and stearic acid (C18:0) in theblended oil, fish oil, soybean oil and yellow grease in the absence ofantioxidants, whereas in the presence of the antioxidants there was noincrease in these fatty acids (data not shown).

TABLE 2 Percentages of selected fatty acids before and after oxidativestress with or without antioxidants. Control ETX + TBHQ Oxidized LSMSEOleic Acid (C18:1) Corn Oil 27.5^(a) 27.6^(a) 28.2^(b) 0.2 Blended22.6^(a) 24.7^(b) 26.5^(c) Oil Fish Oil 9.8^(a) 9.8^(a) 10.8^(b) Soybean27.8^(a) 27.9^(a) 30.5^(b) Oil Yellow 30.3^(a) 30.4^(a) 34.1^(b) GreaseLinoleic Acid (C18:2) Corn Oil 57.7^(a) 57.7^(a) 56.7^(b) 0.3 Blended33.4^(a) 32.8^(a) 30.6^(b) Oil Fish Oil 1.4 1.5 1.4 Soybean 50.2^(a)49.7^(a) 46.1^(b) Oil Yellow 40.6^(a) 40.7^(a) 34.4^(b) Grease LinolenicAcid (C18:3) Corn Oil 1 1 0.9 0.3 Blended 2.7^(a) 2.2^(ab) 1.9^(b) OilFish Oil 3.0^(a) 2.0^(b) 2.1^(b) Soybean 6.1^(a) 6.1^(a) 4.8^(b) OilYellow 5.0^(a) 5.0^(a) 3.2^(b) Grease ^(a,b,c)Means with differentletters within a row are significantly different (P < 0.05).

The level of the highly unsaturated omega-3 unsaturated fatty acid,docosahexaenoic acid (DHA; C22:6) was significantly reduced in oxidizedfish oil (5.4% total fat). The presence of antioxidants, however,prevented this reduction (7.4% total fat vs. 7.5% total fat in thecontrol). The concentrations of all omega 3 and omega 6 fatty acids werereduced in the oxidized blended oil/fat, and the antioxidants partiallyreversed oxidation (FIG. 4). Likewise the concentrations of the omega-3fatty acids, eicosapentaenoic acid, (EPA, C20:5) and DHA were reduced inthe blended oil/fat during oxidative stress, and the antioxidantsreduced the magnitude of the loss (FIG. 5).

These data indicate that the antioxidant combination of ethoxyquin andTBHQ significantly increased the ability of certain oils and fats towithstand extreme oxidative stress. These antioxidants maintained orsignificantly reduced the oxidation of unsaturated fatty acids, such aslinolenic, and linoleic acid, and the highly unsaturated omega 3, andomega 6 fatty acids, and in particular, EPA and DHA.

Example 3 The Combination of Ethoxyquin and TBHQ Prevent the Oxidationof Fatty Acids in Solid Feedstuffs

Approximately 50% of the dietary lipids of cattle come from feedstuffsother than supplemental oils and fat. Feedstuffs such as cottonseed,distillers grains, soybean products and fishmeal significantlycontribute to the total dietary lipids. Most of the lipids from theseingredients contain high levels of unsaturated fatty acids that areprone to oxidation. For example, distillers grains from the ethanolindustry are sources of unsaturated fatty acids that are fairlyunstable. The heating process during distillation and high water contentof the wet distillers grains exacerbate the oxidation process of theunsaturated fatty acids. The end result is a highly oxidized andunstable fat in the distiller grains. The objective of this study was todetermine the effectiveness of the combination of ETX+TBHQ to stabilizethe lipids in wet distillers grains (WDG) or flax seed.

Methods. The oxidative stability of fats in WDG or flax seed wasmeasured in the presence or absence of the blend of ethoxyquin and TBHQ(AGRADO PLUS®) using the active oxygen method (AOM), essentially asdescribed in Example 1. The liquid formulation of antioxidants contained65% ETX, 7% TBHQ, 7% citric acid, 19% propylene glycol, and 2% corn oil.The dry formulation of antioxidants contained 50% ETX, 5% TBHQ, 5%citric acid, and 10% calcium carbonate.

Results. The oxidation of fatty acids in flax seeds was reduced in thepresence of ETX+TBHQ (FIG. 6A). Likewise, the combination of ETX+TBHQreduced the formation of peroxides and hydroperoxides in wet distillersgrains (FIG. 6B).

The fatty acid profile was analyzed in wet distiller grains, asdescribed in Example 1, before and after oxidation in the absence orpresence of the antioxidant blend. The concentration of linoleic acid(C18:2) and linolenic acid (C18:3) decreased during oxidative stress,but the blend of antioxidants prevented this reduction (FIG. 7).

The energy content of grains oxidized in the absence or presence of theantioxidants was measured by bomb calorimetry. For this, the sample washeated to 90° C. and oxygen was bubbled through the sample. The energycontent was reduced 35% relative to a fresh lipid sample, but the energyvalue was maintained in the presence of the antioxidants. The protectiveeffect of antioxidants is critical because oxidation not only reducesthe energy and biological value of oils and fats, but also propagatesthe oxidation of other lipid-based ingredients, such as vitamins andpigments. Thus, these findings indicate that a combination of ethoxyquinand TBHQ can be added to the final diet of dairy and beef cattle toprevent the oxidation of fatty acids and vitamins in feedstuffs.

Example 4 Nutrient Digestion During Ruminal Fermentation Using Fresh orOxidized Fat Diets with or without Antioxidants

The impact of free radicals and oxidative stress on ruminalmicroorganisms is unknown. The objective of this study was to evaluatethe effect of feeding fresh fat or oxidized fat in the absence orpresence of dietary antioxidants on nutrient digestibility usingcontinuous culture fermenters.

Experimental Diets. A lactating dairy ration was formulated to support40 kg/d of milk production with a predicted daily milk index of 24 kg/d.Dietary ingredients and nutrient composition are shown in Table 3. Thediet consisted of 52% forage and 48% concentrate mixture that contained3% experimental fat on a dry mass basis. The experimental fat consistedof a blend of non-stabilized unsaturated fats; the blend contained 33%fish oil, 33% corn oil, 26% soybean oil, and 7% inedible tallow. Half ofthe experimental fat was oxidized by bubbling air through the fat at 92°C. for 24 h to achieve a peroxide value of 215 meq/kg (Table 4).

TABLE 3 Ingredient and nutrient composition (%) of the experimentaldiets. Treatments¹ Items FF FF + AO OF OF + AO Ingredients: AlfalfaHaylage 4.56 4.56 4.56 4.56 Corn Silage 28.12 28.12 28.12 28.12 MixedGrass Hay 19 19 19 19 Soybean Meal 44 15.58 15.58 15.58 15.58 CornGluten Meal 0.66 0.66 0.66 0.66 Soy Hulls 3.8 3.8 3.8 3.8 Flaked Barley5.22 5.22 5.22 5.22 Steam Flaked Corn 17.48 17.48 17.48 17.48 Fresh Fat3.00 3.00 0.00 0.00 Oxidized Fat 0.00 0.00 3.00 3.00 Urea 0.66 0.66 0.660.66 Magnesium Oxide 0.01 0.01 0.01 0.01 Dicalcium Phosphate 0.28 0.280.28 0.28 Sodium Bicarbonate 0.97 0.97 0.97 0.97 Limestone 0.28 0.280.28 0.28 TMin Salt 0.19 0.19 0.19 0.19 ADE Mix 0.11 0.11 0.11 0.11Vitamin E 0.06 0.06 0.06 0.06 Antioxidant Blend 0 0.01 0 0.01 Nutrients:Crude Protein 18.3 18.6 18.8 18.5 Soluble Protein 35.7 31.5 34.8 35.6 (%crude protein) Neutral Detergent Fiber 28.3 28.6 27.2 29 Acid DetergentFiber 18.1 18.4 18 17.9 Nonstructural Carbohydrate 31.8 31.1 31.9 31.6(starch + sugar) Starch 25.8 25.2 26 25.7 Sugar 6 5.9 5.9 5.9 EtherExtract 5.6 5.5 5.4 5.5 Ash 6.2 6 5.8 6.2 Calculated Non-Fiber 41.6 41.442.8 41 Carbohydrate C12:0 0.09 0.10 0.10 0.10 C14:0 1.23 2.10 2.16 1.16C14:1 0.03 0.00 0.00 0.06 C15:0 0.18 0.17 0.19 0.17 C16:0 14.96 14.8715.35 15.36 C16:1 2.36 2.50 2.54 2.53 C18:0 3.37 3.29 3.41 3.46cis-C18:1 18.87 18.84 19.26 19.27 C18:2 38.57 38.87 38.65 37.94 C18:35.08 5.11 5.08 4.99 C20:0 0.53 0.54 0.54 0.56 C21:0 0.45 0.45 0.36 0.38cis-9, trans-11 C18:2 (CLA)² 0.16 0.14 0.12 0.13 C22:0 0.40 0.36 0.400.42 C20:4 0.19 0.20 0.16 0.17 C20:5 2.74 2.81 2.14 2.21 C24:0 0.27 0.440.17 0.16 C22:6 1.54 1.55 1.07 1.13 Other Fatty Acids 8.98 7.66 8.319.79 Total Fatty Acids 4.51 4.44 4.12 4.49 ¹Treatments: FF = fresh fat;FF + AO = fresh fat with antioxidant; OF = oxidized fat; OF + AO =oxidized fat with antioxidant. ²Conjugated linoleic acid

There were four different diet treatments: a) 3% fresh non-oxidized fatwithout added antioxidants (FF−AO); b) 3% fresh non-oxidized fat plus100 mg/kg of dietary antioxidant (FF+AO); c) 3% oxidized fat withoutadded antioxidants (OF−AO); and d) 3% oxidized fat plus 100 mg/kg ofdietary antioxidant (OF+AO). The dietary antioxidant (AO) consisted of aliquid blend of 65% ethoxyquin, 7% TBHQ, 7% citric acid, 19% propyleneglycol, and 2% corn oil. The dietary antioxidant was added to theexperimental fat just prior to mixing of the diets and was added at arate of 100 mg/kg. The diets were stored at 0° C. between feedings, andallowed to come to room temperature prior to feeding. Peroxide valuesand changes in the fatty acid profile (Table 4) were used to assess thequality and stability of the two experimental fats prior to adding theantioxidant.

TABLE 4 Peroxide values and fatty acid profiles of the fresh andoxidized fats. Fresh Fat (FF) Oxidized Fat (OF) Peroxide value (meq/kgfat) 3.5 215 C14:0 (%) 3.6 3.8 C16:0 (%) 14.6 15.8 C18:0 (%) 4.0 4.3C18:1 (%) 21.7 22.9 C18:2 (%) 35.0 34.5 C18:3 (%) 3.6 3.1 C20:5 (%) 5.13.6 C22:6 (%) 2.4 1.7 Omega 6 Total (%) 35.96 34.97 Omega 3 Total (%)12.38 9.98

Continuous Culture System. A twelve-unit dual effluent continuousculture system as described by Hoover et al. (J. Animal Sci. (1976)43:528-534) was used. Ruminal inoculum was obtained from tworumen-cannulated lactating Holstein cows. The two samples were pooledbefore inoculating the 1,164-mL fermenters. Fermenters were fed theexperimental diets (ground to pass a 4-mmm sieve) automatically for 10days in two daily equal feedings at 12-h intervals. The artificialsaliva of Weller and Pilgrim (British J. Nutr. (1974) 32:341-350) wascontinuously infused to provide a liquid dilution rate of 12% per hourover the culture period. All treatments were fermented in triplicate forten days in continuous cultures. Continuous culture conditions were asfollows: liquid dilution rate: 12%/hr, solids retention time: 24 hr,feed intake 100 g dry mass/day, fermentation temperature 39° C., pH wasrecorded at 0.5 hr intervals. The first 7 days were for equilibration.During the last 3 days, the effluents were collected in an ice batch anda 1-L sample was composited and saved for analysis. After the finaleffluent was collected on day 10, the contents of the fermenters wereallowed to settle and the upper fluid layer was used for microbeanalysis.

Chemical Analysis. The feed dry mass was determined by oven drying at100° C. for 24 h. Effluent dry mass was determined by centrifuging a 34to 40 g sample of effluent at 30,000×g for 45 min (Lean et al. (2005) J.Dairy Sci. 88:2524-2536). For digestibility determination, dry matterdigested (DMD) and organic matter digested (OMD) were corrected formicrobial dry matter and organic matter. Neutral detergent fiber contentand acid detergent fiber content in the feed and in continuous cultureeffluents were determined using standard procedures (Goering and VanSoest (1991) Agric. Handbook No. 379, ARS, USDA; Crawford (1983) J.Dairy Sci. 66:1881-1890). Total nitrogen in feed, effluents, andbacterial, ammonia, and ether extraction was determined using standardmethods. Volatile fatty acids were analyzed by gas chromatography (Leanet al. (2005) J. Dairy Sci. 88:2524-2536). Effluent and bacterialconcentration of purines were determined by the procedures of Zinn andOwens (Can. J. Anim. Sci. (1986) 66:157-166). The sugars and starches ofthe feeds and effluents were determined by the procedure of Smith(Wisconsin Agric. Exp. Stn. Res. (1969) Rep. 41), except thatferricyanide was used to detect reducing sugars.

Fermenter outflow samples were freeze-dried and converted to methylesters in sodium methoxide/methanolic HCl as described by Kramer et al.(Lipids (1997) 32:1219-1228). Fermenter outflow fatty acids wereanalyzed on a HP5890A GC (Agilent Technologies, Inc) equipped with aflame ionization detector and a 30 m×0.25 mm (0.2 μm film) Supelco 2380fused silica capillary column. The injector and detector temperatureswere held at 250° C. and 260° C., respectively. The carrier gas was He(20 cm/s) with an inlet pressure of 104 kPa. The column temperature wasprogrammed for 140° C. for 3 min, then increased to 220° C. at 2°C./min, and held at 220° C. for 2 min. Peaks were quantified bycomparison to an internal standard (C17:0).

Statistical Analysis. Data were analyzed as a completely randomizeddesign by analysis of variance using the GLM procedure of SAS (SASInstitute, 2003). Main effects of type of fat and presence of dietaryantioxidant were tested as a 2×2 factorial arrangement. Significantdifferences were declared at P-values less than 0.05 and trends atP-values less than or equal to 0.1 and higher than 0.05.

Results. The nutrient digestibility was analyzed and the results areshown in Table 5; significant values are bolded. The digestion of crudeprotein was reduced in the oxidized fat diet relative to the fresh fatdiet, (P<0.01). The presence of antioxidants restored the digestion ofcrude protein in the oxidized fat diet, however. Although digestion ofthe neutral detergent fiber was not affected by fat source, theantioxidants significantly increased the digestion of the neutraldetergent fibers in both the fresh and oxidized fat diets (P<0.02).Digestion of the acid detergent fiber tended to be greater in theoxidized fat diet relative to the fresh fat diet (P<0.08). Again, theaddition of the antioxidants to either fat source increased thedigestion of the acid detergent fiber (P<0.04). These findings suggestthat addition of antioxidants reduced the negative impact of fats onfiber digestion.

TABLE 5 Nutrient digestibility of the different diets. Treatments¹P-values Item FF − AO OF − AO FF + AO OF + AO Fat AO Fat × AO Digestion,% Dry Matter 67.4 69.8 69.5 70.9 0.49 0.56 0.86 Organic Matter 61.0 61.865.0 63.1 0.76 0.17 0.47 Crude Protein 97.9 87.5 98.5 93.0 0.01 0.220.33 Neutral Detergent Fiber 35.7 40.1 46.0 45.2 0.51 0.02 0.35 AcidDetergent Fiber 44.0 50.0 50.9 54.0 0.08 0.04 0.53 Nonstructural 69.570.7 69.4 69.0 0.72 0.43 0.45 Carbohydrate (starch + sugar) TotalCarbohydrate 32.2 33.5 34.8 34.9 0.47 0.05 0.534 digested/day (g/d)¹Treatments: FF = fresh fat; FF + AO = fresh fat with antioxidant; OF =oxidized fat; OF + AO = oxidized fat with antioxidant

Analysis of the volatile fatty acids revealed an increased production ofbutyric acid in the oxidized fat diet with and without antioxidants(Table 6). There were no differences in the production rate or molarratio of any other volatile fatty acid (Table 6). Likewise, the averagefermentation pH did not change under any treatment.

TABLE 6 Volatile Fatty Acid and pH Analyses. Treatments¹ P-values ItemFF − AO OF − AO FF + AO OF + AO Fat AO Fat × AO Total Volatile FattyAcids 385 397 396 386 0.87 0.99 0.22 (mmoles/d) Acetic Acid 205 206 207209 0.76 0.61 0.99 Propionic Acid 109 114 115 104 0.62 0.70 0.22Isobutyric Acid 3.1 3.0 2.7 2.9 0.93 0.04 0.19 Butyric Acid 53 59 52 580.02 0.66 0.97 Molar %: Acetic Acid 53.3 52.0 52.4 54.0 0.85 0.57 0.17Propionic Acid 28.4 28.8 29.1 26.9 0.46 0.62 0.30 Isobutyric Acid 0.790.74 0.69 0.74 0.99 0.14 0.16 Butyric Acid 13.8 14.8 13.2 15.0 0.02 0.610.39 A-P Ratio 1.89 1.82 1.81 2.02 0.52 0.61 0.24 Average pH 6.29 6.146.15 6.18 0.27 0.32 0.10 ¹Treatments: FF = fresh fat; FF + AO = freshfat with antioxidant; OF = oxidized fat; OF + AO = oxidized fat withantioxidant.

Example 5 Microbial Efficiency During Ruminal Fermentation Using Freshor Oxidized Fat Diets with or without Antioxidants

The objective of this study was to evaluate the effect of feeding freshfat or oxidized fat in the absence or presence of dietary antioxidantson microbial efficiency, as monitored by nitrogen metabolism, duringruminal fermentation using continuous culture fermenters.

Methods. The experimental diets, continuous culture system, and chemicalanalyses were as described in Example 4.

Results. The effects of the different treatments on nitrogenpartitioning and microbial efficiency were analyzed and the results areshown in Table 7, with the significant values bolded. Microbial growthwas significantly reduced in the presence of the oxidized fat dietrelative to the fresh fat diet (P<0.03). By-pass nitrogen, however, wassignificantly increased with the oxidized fat diet, resulting in highernon-ammonia nitrogen levels in the oxidized fat treatment. The additionof antioxidants increased microbial growth under both fresh and oxidizedfat diets (P<0.09). Consequently, the fresh fat diet with antioxidantsresulted in more microbial nitrogen than any other treatment. Additionof antioxidants reduced ammonia levels under both fat treatments(P<0.06).

TABLE 7 Effect of treatments on nitrogen partitioning, microbial growthand microbial efficiency. Treatments¹ P-values Item FF − AO OF − AO FF +AO OF + AO Fat AO Fat × AO Crude Protein Digested, % 97.9 87.5 98.5 93.00.01 0.22 0.34 Non-ammonia N, g/d 2.59 2.69 2.68 2.69 0.01 0.01 0.01Ammonia N, mg/d 18.93 17.98 17.58 16.62 0.16 0.06 0.99 ByPass N, g/d0.07 0.41 0.05 0.23 0.01 0.21 0.31 Microbial N, g/d 2.52 2.28 2.63 2.470.03 0.09 0.66 Efficiencies: Mic.N/DMD² 37.4 32.7 38.1 34.8 0.03 0.390.63 Mic.N/OMD³ 43.8 39.2 43.1 41.8 0.10 0.54 0.32 Mic.N/CHOD⁴ 78.4 68.275.7 71.0 0.08 0.99 0.48 Feed N, %⁵ 79.7 78.4 81.3 81.0 0.26 0.01 0.49TVFA/CHOD⁶ 12.01 11.88 11.38 11.08 0.53 0.06 0.80 TVFA/Mic.N⁷ 153 175150 158 0.05 0.16 0.30 ¹Treatments: FF = fresh fat; FF + AO = fresh fatwith antioxidant; OF = oxidized fat; OF + AO = oxidized fat withantioxidant. ²Grams microbial nitrogen (Mic.N) produced per kg drymatter digested (DMD). ³Grams microbial nitrogen (Mic.N) produced per kgtotal organic matter digested (OMD). ⁴Grams microbial nitrogen ((Mic.N)produced per kg total carbohydrate digested (CHOD). ⁵Digested feednitrogen (N) converted to microbial nitrogen, %. ⁶Moles total volatilefatty acids (TVFA) produced per kg carbohydrate digested (CHOD). ⁷Molestotal volatile fatty acids (TVFA) produced per kg microbial N (Mic.N)produced.

Microbial efficiencies were high across all treatments, possibly due tothe low amount of carbohydrate digested (see Table 5) and the overallhigh yield of microbial nitrogen. Compared to the oxidized fat diet,however, the fresh fat diet resulted in somewhat higher microbialnitrogen/unit of digested dry matter, organic matter, and totalcarbohydrate (Table 7). Although not statistically significant, additionof antioxidants to the oxidized fat diet improved microbial efficiency.Incorporation of feed nitrogen into microbial nitrogen was significantlyincreased in the presence of antioxidants regardless of the fat source.Unlike the positive effects of antioxidants on ammonia levels, feednitrogen efficiency, and microbial growth, antioxidants decreased thenitrogen content of the microbes grown with both fat sources (P<0.02)(Table 8).

TABLE 8 Effects of treatments on the composition of the microbes.Treatments¹ P-values Item FF − AO OF − AO FF + AO OF + AO Fat AO Fat ×AO Nitrogen, % 9.89 9.73 9.68 9.48 0.05 0.02 0.81 Ash, % 8.76 10.19 9.0313.71 0.13 0.33 0.39 RNA-nitrogen, % of total 10.27 10.83 10.09 10.470.24 0.49 0.81 nitrogen ¹Treatments: FF = fresh fat; FF + AO = fresh fatwith antioxidant; OF = oxidized fat; OF + AO = oxidized fat withantioxidant.

Example 6 Metabolism of Fatty Acids During Ruminal Fermentation UsingFresh or Oxidized Fat Diets with or without Antioxidants

The objective of this study was to evaluate the effect of feeding freshfat or oxidized fat in the absence or presence of dietary antioxidantson fatty acid metabolism during ruminal fermentation using continuousculture fermenters.

Methods. The experimental diets, continuous culture system, and chemicalanalyses were as described in Example 4.

Results. Oxidation of the experimental fat by bubbling air duringheating oxidized the long chain unsaturated fatty acids as reflected bythe lower concentration of EPA (C20:5) and DHA (C22:6) and higher levelsof peroxides in the oxidized fat relative to the fresh fat (see Table 4above). Differences in the fatty acid profile of the two types of fatwere reflected in the total amount of fat and the concentration of fattyacids in the final diets. The oxidized fat diet contained lowerconcentrations of long chain fatty acids such as C21:0, CLA, C20:4,C20:5, C24:0, and C22:6 than the fresh fat diet (see Table 3 above).

The outflow of fatty acids in the effluent varied with type of fat dietand presence or absence of antioxidants as detailed in Table 9.Fermenters fed fresh fat diets had lower outflow of C16:0 (P<0.02),C18:0 (P<0.01), C22:0 (P<0.08), and C24:0 (P<0.007), and higher outflowof trans-C18:1 (P<0.05), EPA (P<0.037), and other unsaturated fattyacids (P<0.012) than fermentors fed oxidized fat diets. In general,fresh fat diets increased the outflow of unsaturated fatty acids(P<0.06) and decreased the outflow of saturated fatty acids (P<0.01)relative to oxidized fat diets. The presence of antioxidants in thediets reduced the outflow of C18:3 (P<0.076) in all diets and reducedDHA outflow in the oxidized fat diets (P<0.01).

TABLE 9 Effect of treatments on daily outflow of fatty acids in effluentof continuous cultures of mixed ruminal microbes. Treatments¹ P-ValuesItem, mg/d FF OF FF + AO OF + AO SE AO Fat AO * Fat C_(16:0) 677.32705.05 664.78 725.72 13.49 0.77 0.01 0.25 C_(16:1) 59.85 53.45 61.3357.64 5.45 0.62 0.38 0.81 C_(18:0) 192.96 299.20 198.63 273.39 26.110.71 0.01 0.56 trans-C_(18:1) 1,408.29 1,235.12 1,447.66 1,366.72 61.000.19 0.07 0.47 Cis-C_(18:1) 380.34 395.03 394.45 426.51 16.77 0.21 0.210.61 C_(18:2) 339.70 340.39 329.08 354.01 20.17 0.94 0.54 0.56 C_(20:0)19.72 19.61 19.45 21.12 0.61 0.34 0.24 0.18 C_(18:3) 62.60 64.39 50.6152.08 6.16 0.08 0.79 0.97 cis-9-trans 17.71 14.38 13.57 10.11 3.51 0.260.36 0.98 11C_(18:2)CLA² C_(22:0) 19.38 21.59 21.14 22.52 0.81 0.14 0.060.63 C_(20:5) 39.71 22.42 36.53 24.69 3.69 0.90 0.01 0.48 C_(24:0) 16.3717.23 16.86 19.87 0.54 0.02 0.01 0.08 C_(22:6) 26.22 34.12 31.75 15.093.68 0.10 0.26 0.01 Other FA³ 846.33 769.03 801.52 810.95 14.49 0.930.05 0.02 Unsaturated 1,963.72 1,774.40 1,982.26 1,890.38 73.39 0.380.09 0.52 FA⁴ Saturated 987.76 1185.39 983.01 1130.53 27.54 0.31 0.010.38 FA⁵ ¹Treatments: FF = fresh fat; FF + AO = fresh fat withantioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.²CLA = Conjugated linoleic acid ³FA = Fatty Acids ⁴Unsaturated FAinclude: C_(14:1), C_(16:1), isomers-C_(18:1), C_(18:2), C_(18:3), CLA,C_(22:1), C_(20:5), and C_(22:6) ⁵Saturated FA include: C_(12:0),C_(14:0), C_(15:0), C_(16:0), C_(18:0), C_(20:0), C_(21:0), C_(22:0),and C_(24:0)

The proportion of dietary fatty acids recovered in the effluents wascalculated by dividing the outflow of fatty acids in the effluent by theamount of fatty acids fed daily (Table 10). The percentage of C16:0(P<0.01), C18:0 (P<0.068), C24:0 (P<0.0001) and the sum of the majorsaturated fatty acids (P<0.001) recovered in the effluent wassignificantly higher in the oxidized fat diet than in the fresh fatdiet. The presence of antioxidants in the diets reduced the proportionof C18:3 (P<0.05) and the sum of the major saturated fatty acids(P<0.033) recovered in the effluents. Significant differences in thepercentage of recovery of several fatty acids were observed in thepresence of antioxidants in the different fat sources. In the presenceof the antioxidants, the recovery of C22:0, DHA and other fatty acidswere reduced in oxidized fat diet, but there was no change (e.g., DHA)or increased recovery (e.g., C22:0 and other fatty acids) in the freshfat diet. However, in the presence of antioxidants, the recovery ofC24:0 was reduced in the fresh fat diet but increased in the oxidizedfat diet.

TABLE 10 Effects of treatments on the recovery of fatty acids in theeffluents of continuous cultures of mixed ruminal microbes. Treatments¹P-values Item g/100 g FF − AO OF − AO FF + AO OF + AO SE AO Fat AO × FatC16:0 100.4 111.6 100.7 105.2 2.0 0.18 0.01 0.14 C16:1 56.3 51.2 55.355.7 4.9 0.89 0.35 0.96 C18:0 126.8 212.9 135.9 175.9 17.4 0.44 0.010.22 cis-C18:1 44.7 49.8 47.2 49.3 2.0 0.63 0.10 0.46 C18:2 19.5 21.419.1 20.8 1.2 0.67 0.17 0.95 C20:0 83.0 88.7 81.3 84.4 2.6 0.28 0.130.62 C18:3 27.3 30.8 22.3 23.2 2.7 0.05 0.45 0.65 cis-9-trans-11- 249.5298.3 211.0 179.3 60.6 0.23 0.89 0.53 C18:2 (CLA) C22:0 106.7 132.5132.1 118.2 4.6 0.26 0.24 0.01 C20:5 (EPA) 32.1 25.4 29.3 24.9 3.1 0.600.12 0.73 C24:0 134.0 246.8 85.9 285.5 3.6 0.23 0.01 0.01 C22:6 (DHA)37.6 77.1 46.3 29.7 5.7 0.01 0.07 0.01 Other Fatty Acids 208.9 224.8235.7 184.5 3.9 0.12 0.01 0.01 Unsaturated 62.6 62.5 63.8 61.5 2.4 0.960.62 0.67 Fatty Acids² Saturated Fatty 97.5 122.3 95.0 110.9 2.7 0.030.01 0.14 Acids³ ¹Treatments: FF = fresh fat; FF + AO = fresh fat withantioxidant; OF = oxidized fat; OF + AO = oxidized fat with antioxidant.²Unsaturated fatty acids include: C14:1, C16:1, isomers-C18:1, C18:2,C18:3, CLA, C22:1, EPA, and DHA. ³Saturated fatty acids include: C12:0,C14:0, C15:0, C16:0, C18:0, C20:0, C21:0, C22:0, and C24:0.

These data indicate that oxidized fat diets lead to reduceddigestibility of crude proteins, decreased microbial protein synthesis,and reduced outflow of unsaturated fatty acids. The addition of theantioxidants, ethoxyquin and TBHQ, reversed or lessened these effects.

Example 7 Effects of Feeding Fresh and Oxidized Fat in the Presence andAbsence of Antioxidants on Dairy Cow Lactation

The objective of this study was to evaluate the effects of feeding freshor oxidized soybean oil in the absence or presence of a combination ofdietary antioxidants on milk production and antioxidant status of cowsduring mid to late lactation. Mid to late lactating heifers were fed oneof four diets for six weeks, during which time milk yield and otherperformance parameters were monitored. Antioxidant status was determinedby measuring the activity of antioxidant enzymes in the blood plasma.

Treatments and experimental design. Forty-four primiparous mid to latelactation Holstein cows housed in a tie-stall barn at Spruce HavenResearch facility (NY) were randomly assigned to treatments at 175 daysin milk (DIM). The study consisted of four treatments: a) freshnon-oxidized soybean oil (FF) added to the diet at 2%; b) freshnon-oxidized soybean oil added to the diet at 2% plus 100 mg/kg ofdietary antioxidant (FF+AO); c) oxidized soybean oil (OF) added to thediet at 2%; and d) oxidized soybean oil added to the diet at 2% plus 100mg/kg of dietary antioxidant (OF+AO). The dietary antioxidant consistedof a liquid blend of ethoxyquin and tertiary butyl hydroquinone (AGRADOPLUS®, Novus International; St. Louis, Mo.). For the first 15 d ofstudy, all cows were fed ad libitum a control diet (Table 11) containing2% fresh non-stabilized soybean oil (FF). During this time, individualdaily milk and feed intake were measured. At the end of this adaptationperiod, body condition score (BCS), body weight (BW), and blood sampleswere taken and served as covariate. The cows were then switched to oneof the four treatment diets (Table 12) for six weeks. Treatmentassignments were balanced for DIM, milk yield during the covariateperiod, and body condition scores. The experimental diet consisted of58% forage and 42% concentrate mixture that contained 2% experimentalfat on a dry mass (DM) basis. The diets were formulating usingCornell-Penn-Minor (CPM) model following NRC 2001 recommendations andcurrent industry practices. The experimental fat consisted ofnon-stabilized soybean oil. Half of the experimental fat was oxidized bybubbling air through the fat at 92° C. for 24 h to achieve a peroxidevalue of 240 meq/kg (method Cd 12-57; AOCS, 1997). The fresh fat had aperoxide value of 0.5 meq/kg. The dietary antioxidant (AO) blend wasadded to the experimental fats just prior to mixing of the diets toachieve 100 mg/kg of final diet on an as fed basis. Fresh and oxidizedsoybean oils were kept frozen prior to two days before feeding.

TABLE 11 Composition of Basal Diet Ingredient % Dry Mass (DM) CornSilage 2004 47.07 Haylage, 2^(nd) cut 2005 10.51 Fine ground corn 10.51Soybean Meal 49 12.61 Corn Distillers 2.18 SoyPlus 1.47 SodiumBicarbonate 0.96 Calcium Carbonate 0.91 Geobond 0.51 Corn Gluten Meal0.43 Fishmeal 0.44 Bloodmeal 0.44 Urea 0.24 Salt 0.27 Magnesium Oxide0.31 MonoCal 21 0.24 Celmanax 0.19 Selenium 270 0.09 Dynamate 0.05Vitamin E 20000 0.06 KeyDyPrmx2.5 0.4 Beet Pulp Pellets 8.40 Soybean oil2.00 Alimet 0.08

TABLE 12 Diet Analysis, % Dry Matter Basis. Treatment Diets Component FFOF FF + AO OF + AO Crude Protein (CP) 17.8 18.0 18.1 18.2 SolubleProtein (% CP) 38.6 43.8 36.3 37.6 Neutral Detergent Fiber (NDF) 31.132.1 31.9 31.5 Acid Detergent Fiber (ADF) 18.8 18.7 19.7 18.8Non-Structural Carbohydrate 29.9 30.9 28.8 30.4 (NSC¹) Starch 24.5 25.523.3 24.9 Sugar 5.4 5.4 5.5 5.5 Ether Extract 4.6 5.2 4.8 4.8 Ash 7.67.6 7.5 7.5 Non-Fiber Carbohydrate (NFC²) 38.9 37.0 37.7 38.0 ¹NSC =starch + sugar ²Calculated NFC

Performance monitoring and sample collection. The amount of feed offeredand the amount of feed not eaten (orts) were recorded daily for eachcow. The am and pm milk weights were recorded daily for each cow. Eachweek, individual milk samples taken during one 24-h period werecomposited based upon the amount of milk produced at each milking andanalyzed for milk protein and fat by infrared spectrophotometry (NYDHIA, Ithaca, N.Y.).

Blood samples were taken every two weeks for evaluation of oxidativestress status. Blood samples were taken from each cow tail vein usingheparin plasma tubes at 2 h after feeding, immediately placed on ice,and then centrifuged at 1,000 g for 10 min. The supernatant plasma wasfrozen for later analysis of superoxide dismutase (SOD) using the assaykit supplied by Cayman Chemical Company (Catalog # 706002; Ann Arbor,Mich.), total antioxidant status (TAS) using the kit supplied byCalbiochem (Catalog # 615700; Darmstadt, Germany), glutathioneperoxidase (GPX) using the assay kit supplied by Cayman Chemical Company(Catalog # 703102; Ann Arbor, Mich.) and malondialdehyde (MDA) usingcalorimetric assay kit supplied by Calbiochem (Darmstadt, Germany).Daily health status of the cows was monitored during entire study.

Dry matter content of the total mixed rations (TMR) was measured weekly.Concentrate and silage samples were taken every two weeks and compositedmonthly, and frozen for later analysis. The source of forage wasmaintained constant during the entire study. Feed samples were analyzedfor crude protein (CP), neutral detergent fiber (NDF), acid detergentfiber (ADF), ethanol as described in Example 4.

Statistics. The study was designed as a completely randomized designwith repeated measurements with a 2×2 factorial treatment arrangement,where the main effects were type of fat (FF vs OF) and the presence orabsence of AO. Data were analyzed as a completely randomized design withrepeated measurements using the MIXED procedure of SAS® (SAS Institute,2003). Week was used in the repeated measurement statement with cowwithin treatment as the error term. Pretreatment measurements were usedduring analysis of covariate. Significance differences were declared atP-values less than 0.05 (bolded in the tables) and trends at P-valuesless than or equal to 0.1 and higher than 0.05.

Results. Analysis of the data revealed few differences due to type offat in the diet (fresh vs. oxidized), but there were significantdifferences between the diets with or without antioxidants. The data inTables 13-15 are presented as means of the treatments withoutantioxidants (−AO), with antioxidants (+AO), with fresh fat (FF), andwith oxidized fat (OF).

Cows fed AO exhibited significant changes in milk production and milkconstituents (Table 13). Cows responded to AO by significantlyincreasing dry matter intake (DMI) (P=0.007), 3.5% fat corrected milk(3.5 FCM) (P=0.01), and milk fat yield (P=0.01), while decreasing milkprotein content (P=0.03) (Table 13). There was a trend towards increasedmilk yield (P=0.08) in the presence of AO. Feeding OF reduced DMI(P=0.04), and increased milk fat yield (P=0.02).

Milk (P=0.0003), 3.5 FCM (P=0.04), DMI (P=0.003) and protein yield(P=0.0001) were gradually reduced, while BW (P=0.0001) and BCS (P=0.02)were improved during the 6-week study as a reflection of the mid to latestage of lactation of the cows used in the trial (175 DIM). Milk fatcontent, milk fat yield, and milk protein content did not change withweek. No dietary treatment by week interaction was observed for any ofthe performance parameters, except for BW. At the end of the trial, cowsfed OF had the lowest BW (P=0.05).

Significant changes in antioxidant status were observed in cows fed AO(Table 14). Cows fed AO showed higher (P<0.002) TAS over cows not fed AOindependently of the type of fat fed. Over time, TAS levels wereimproved with AO but reduced in its absence (P=0.0009). Type of fat didnot affect plasma TAS but a significant week by AO by type of fat effectwas observed (P=0.003). By the end of the trial, cows fed AO+OF showedthe most improved TAS (0.42±0.04 mM) whereas cows fed OF in the absenceof AO showed the lowest TAS values (0.07±0.04 mM).

TABLE 13 Effects of treatment on milk production and milk constituents.Treatments¹ P-values −AO +AO FF OF SE² AO Fat AO × Fat Week Items Milk,kg/d 27.38 28.12 27.79 27.71 0.29 0.08 0.84 0.92 0.0003 FCM, kg/d 27.3228.36 27.59 28.10 0.28 0.01 0.202 0.26 0.04 DMI, kg/d 20.28 20.91 20.8320.36 0.16 0.0073 0.04 0.35 0.0029 BW, kg 627.67 631.40 632.42 626.653.55 0.458 0.255 0.98 0.0001 BCS 3.64 3.57 3.60 3.61 0.04 0.076 0.9040.75 0.02 Milk constituents: Fat % 3.49 3.56 3.48 3.57 0.04 0.25 0.140.14 0.27 Fat yield, kg/d 0.95 1.00 0.95 1.00 0.02 0.01 0.02 0.24 0.22Protein % 3.03 2.96 3.00 2.99 0.03 0.03 0.68 0.15 0.15 Protein yield,kg/d 0.82 0.83 0.83 0.83 0.01 0.57 0.92 0.12 0.001 ¹Treatments: FF =fresh fat; +AO = with antioxidant; OF = oxidized fat; −AO = withoutantioxidant ²Standard error

TABLE 14 Effects of Treatments on Plasma Parameters. P-valuesTreatments¹ Week × Week × Items: −AO +AO FF OF SE² AO Fat AO × Fat WeekWeek × Fat AO AO × Fat TAS³, mM 0.17 0.24 0.21 0.20 0.01 0.002 0.53 0.270.04 0.23 0.0009 0.003 MDA⁴, uM 10.03 10.21 9.66 10.58 0.43 0.76 0.1440.69 0.05 0.68 0.17 0.03 GPX⁵, nmol/mg 50.91 75.57 57.43 69.06 4.4 0.0090.05 0.81 0.04 0.03 0.21 0.25 protein SOD⁶, U/mg 0.023 0.023 0.021 0.0250.008 0.549 0.0007 0.047 0.125 0.203 0.529 0.758 protein ¹Treatments: FF= fresh fat; +AO = with antioxidant; OF = oxidized fat; −AO = withoutantioxidant ²Standard error ³TAS = total antioxidant status in plasma⁴MDA = malondialdehyde in plasma ⁵GPX = Gluthathione peroxidase activity⁶SOD = Superoxide dismutase in plasma

MDA values in plasma were reduced from 10.98 to 9.54±0.46 μM as theweeks on trial progressed (P=0.005). A significant week by type of fatby AO effect (P=0.03) was observed. Cows fed the OF diet without AOshowed the highest MDA levels at 2 weeks (13.27±0.9 μM) and 6 weeks(10.9±0.9 μM) on trial when compared to the rest of the treatments.

Plasma GPX activity increased when AO was added to the diet (P=0.009)and when feeding OF vs FF, but this effect was only observed by the endof the trial at six weeks (P=0.07). Plasma SOD activity increased whenfeeding OF (P=0.0007) and was the highest when feeding OF+AO (P=0.05).

Conclusions. Feeding OF reduced intake without compromising milk yieldbut increased milk fat yield. Feeding OF for 6 weeks in the absence ofAO increased plasma MDA and GPX activity, but reduced plasma antioxidantstatus of the cow. Feeding AO reversed many of these effects andprovided additional benefits. Feeding AO not only improved dry matterintake, milk yield, milk fat yield and fat corrected milk, but alsoimproved plasma antioxidant status and antioxidant enzyme activityindependent of the degree of oxidation of the fat fed to the cows.

1. A method for increasing milk fat in a ruminant animal, the methodcomprising: (a) adding to a final diet a first antioxidant, the firstantioxidant being a quinoline compound, and a second antioxidant that isdifferent than the first antioxidant; and (b) feeding to a ruminantanimal the final diet comprising the first antioxidant and the secondantioxidant, wherein the feeding of the first and second antioxidant tothe ruminant animal increases milk fat in the ruminant animal.
 2. Themethod of claim 1, wherein the first antioxidant is a substituted1,2-dihydroquinoline and the second antioxidant is selected from thegroup consisting of a synthetic antioxidant, a semi-syntheticantioxidant, and a natural antioxidant.
 3. The method of claim 2,wherein the substituted 1,2-dihydroquinoline is a compound comprisingformula (I):

wherein: R¹, R², R³ and R⁴ are independently selected from the groupconsisting of hydrogen and an alkyl group having from 1 to about 6carbons; and R⁵ is an alkoxy group having from 1 to about 12 carbons. 4.The method of claim 3, wherein the substituted 1,2-dihydroquinoline is6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline.
 5. The method of claim 1,wherein the first antioxidant is6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and the second antioxidantis tertiary butyl hydroquinone.
 6. The method of claim 1, wherein thefirst antioxidant and second antioxidant are formulated as a drycomposition.
 7. The method of claim 6, wherein the dry compositioncomprises from about 30% to about 70% by weight of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and from about 1% to about10% by weight of tertiary butyl hydroquinone.
 8. The method of claim 6,wherein the dry composition comprises from 45% to-55% by weight of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline and from 3% to 7% byweight of tertiary butyl hydroquinone.
 9. The method of claim 1, whereinthe first antioxidant and the second antioxidant are fed to the ruminantin its water supply.
 10. The method of claim 1, wherein the firstantioxidant and the second antioxidant are fed to the ruminant as a partof its feed ration.
 11. The method of claim 1, wherein the firstantioxidant and the second antioxidant are fed to the ruminant animalsimultaneously as a part of the same composition.
 12. The method ofclaim 1, wherein the first antioxidant and the second antioxidant arefed to the ruminant animal sequentially as a part of separatecompositions.
 13. The method of claim 1, wherein the ruminant is fed afat source comprising from about 1% to about 10% on a dry mass basis ofthe ruminant feed ration.
 14. The method of claim 13, wherein the fatsource is a non-inert fat selected from the group consisting of plantderived fat, fish derived fat, animal derived fat, oilseeds, distillersgrains, and combination thereof.
 15. The method of claim 13, wherein thefat source comprises unsaturated fatty acids.
 16. The method of claim 1,wherein the ruminant animal is selected from a beef cow, a dairy cow anda sheep.
 17. The method of claim 1, wherein the ruminant animal is adairy cow, the first antioxidant is6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, and the secondantioxidant is tertiary butyl hydroquinone.
 18. The method of claim 17,wherein the dairy cow is fed from about 20 to about 250 ppm of6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline as a part of its feedration on an as fed basis.
 19. The method of claim 1, wherein thefeeding to a ruminant animal the final diet further comprises feeding anoxidized fat or fresh fat to the ruminant animal.
 20. The method ofclaim 6, wherein the feeding to a ruminant animal the final dietcomprising the first antioxidants, second antioxidant and fat improves aplasma antioxidant status and an antioxidant enzyme activity of theruminant animal.
 21. The method of claim 7, wherein the improvement inplasma antioxidant status and antioxidant enzyme activity of theruminant animal is independent of the degree of oxidation of the fat fedto the ruminant animal.
 22. The method of claim 1, further comprisingmeasuring the milk fat of the ruminant animal.